The present invention relates to the field of antisense pharmaceuticals, and more specifically to methods for reducing the immunostimulatory response which may be induced in treated individuals by such antisense pharmaceuticals.
Antisense oligonucleotide technology presents an exciting new therapy for many diseases, including pathogenic infections, cancer, and inherited conditions. The field has progressed enormously over the past decade, and currently numerous clinical trials are in progress or are proposed. Antisense oligonucleotides act by binding to a target nucleic acid by Watson-Crick or Hoogsteen base-pairing. Antisense oligonucleotides may be designed to target and to inhibit any single gene within an organism""s genome. For example, the oligonucleotides of SEQ ID NO:1 and SEQ ID NO:2 are phosphorothioate oligonucleotides complementary to the gag and rev regions of HIV-1 which inhibit HIV-1 replication, and the phosphorothioate oligonucleotide of SEQ ID NO:3 binds to the human p53 oncogene. The antisense approach is currently the only known strategy that has broad potential for precise and effective modulation of the expression of specific genes in a disease situation.
However, some antisense oligonucleotides containing phosphorothioate linkages exhibit an immunostimulatory response, causing B cell proliferation and/or an antibody response both in vitro and in vivo. This immunostimulatory response is not characteristic of all antisense oligonucleotides containing phosphorothioate linkages. For example, it is known that the phosphorothioate oligonucleotide of SEQ ID NO:3 does not induce an immunostimulatory effect.
Phosphorothioate oligonucleotide immunostimulatory effects appear to be dependent on particular sequences within the oligonucleotide but remain independent of whether the oligonucleotide is antisense, sense, or scrambled with respect to the respective target gene. Some phosphorothioate oligonucleotides induce cell proliferation, and other phosphorothioate oligonucleotides produce no immunostimulatory effect at all. McIntyre et al. (1993) Antisense Res. Dev. 3:309-322 discloses that certain oligonucleotides can cause pronounced splenomegaly in athymic nude mice. Messina et al. (1993) Cell Immunol. 147:148-157; and Pisetsky et al. (1994) Life Sciences 54:101-107 disclose that DNA as well as structurally related synthetic oligonucleotides and polynucleotides stimulate lymphocytes, but the mechanism for this stimulation is still not fully understood. B cells are usually activated from the resting state by antigen binding to surface immunoglobulin. In mice, activation can also be modulated by physiological mediators, such as interleukin-2 (IL-2), interleukin-4 (IL-4), xcex3-interferon, and non-physiological mitogens, such as lipopolysaccharide (LPS), Concanavalin A (con A), and pokeweed mitogen (PWM).
Certain sequence motifs or structures of oligonucleotides may play important roles in causing stimulation of murine cells. Kuramoto et al. (1992) Jpn. J. Cancer Res. 83:1128-1131 discloses that the presence of particular palindromic sequences including 5xe2x80x2-CG-3xe2x80x2 motif(s) is a critical determinant in oligonucleotides for induction of natural killer cell activation and interferon production. Krieg et al. (1995) Nature 374:546-549 discloses that optimal B cell activation requires a DNA motif in which an unmethylated CpG dinucleotide is flanked by two 5xe2x80x2-purines and two 3xe2x80x2-pyrimidines.
Because of the continued need for specific treatments for diseases and inherited conditions, and the high level of specificity provided through use of antisense therapeutics capable of modulating the expression levels of targeted genes, a need exists for reducing the immunostimulatory response induced by certain phosphorothioate oligonucleotides.
Some cyclodextrins, also known as cycloamyloses, and various substituted derivatives thereof, such as hydroxypropyl-, hydroxyethyl-, methyl-, or sulfate-substituted cyclodextrins, have the ability to enhance the solubility and availability of a variety of pharmacological agents. For example, 2-hydroxypropyl xcex2-cyclodextrin (HPCD) substantially enhances solubility and uptake of some sparingly soluble drugs such as hydrophobic protein containing drugs (Brewster et al. (1991) Pharmaceut. Res. 8:792-795; Yaksh et al. (1991) Life Sci. 48:623-633) such as insulin (Merkus et al. (1991) Pharmaceut. Res. 8:588-592), bovine growth hormone (Simpkins et al. (1991) J. Parenteral Sci. Technol. 45:266-269), and methyltestosterone (Muller et al. (1991) J. Pharmaceut. Sci. 80:599-604). In addition, ethylated-xcex2-cyclodextrin has been used as slow-release type carriers for hydrophilic drugs such as diltiazem (Horiuchi et al. (1990) J. Pharmaceut. Sci. 79:128-132). Cyclodextrins have also been found to eliminate some of the undesirable side-effects of the drugs to which they have been complexed. For example, when used as a vehicle for rectal absorption, HPCD can suppress the local irritation of certain drugs (Arima et al. (1992) J. Pharmaceut. Sci. 81:1119-1125).
Other cyclodextrins have unique biological features. For example, cyclodextrin sulfates have anti-inflammatory, antilipemic, and antiviral activity, and have been found to inhibit replication of HIV by either prevention of viral absorption or budding (Pitha et al. (1991) J. Pharmaceutic. Res. 8:1151-1154; Anand et al. (199) Antiviral Chem. Chemother. 1:41-46); Moriya et al. (1991) J. Med. Chem. 34:2301-2304; Weiner et al. (1992) Pathobiol. 60:206-212). In addition, cyclodextrin sulfates have protective effects on the gentamicin-induced nephrotoxicity (Uekama et al. (1993) J. Pharm. Pharmacol. 45:745-747) and on hemolysis of erythrocytes (Weisz et al. (1993) Biochem. Pharmacol. 45:1011-1016).
Since cyclodextrins are biocompatible polymers composed of naturally occurring D-glucose subunits, their therapeutic application has been regarded are relatively safe. Indeed, in vivo administration of cyclodextrin concentrations of 5 to 10% has been generally used to enhance adsorption of drugs in animal studies, and no significant cytotoxic effects have been reported. (Gerloczy et al. (1994) J. Pharmaceut. Sci. 83:193-196). Besides standard intravenous administration, cyclodextrins can be easily absorbed through nasal (Merkus et al. (1991) Pharm. Res. 8:588-592; Shao et al. (1992) Pharm. Res. 9:1157-1163), intestinal (Nakanishi et al. (1992) Chem. Pharm. Bull. 40:1252-1256), corneal (Jansen et al. (1990) Lens Eye Tox. Res. 7:459-468), rectal epithelium (Arima et al. (1992) J. Pharm. Soc. Japan 112:65-72) routes, and by transdermal injection (Yoshida et al. (1990) Chem. Pharm. Bull. 38:176-179).
However, cyclodextrins have not been known to alter the effects of drugs which stimulate the mammalian immune system.
The present inventors have discovered a method of reducing the immunostimulatory effects of certain phosphorothioate oligonucleotides by adding a cyclodextrin to the pharmaceutical formulation containing the phosphorothioate oligonucleotide
In one embodiment, the invention provides a method of reducing an immunostimulatory response of a mammal to a phosphorothioate oligonucleotide which comprises the steps of administering a therapeutic formulation containing the phosphorothioate oligonucleotide and at least one cyclodextrin to the mammal; and monitoring the immune response of the mammal.
In another embodiment, the invention provides a method of reducing an immunostimulatory response of a mammal to a protein which comprises the steps of administering a therapeutic formulation containing the protein and at least one cyclodextrin to the mammal; and monitoring the immune response of the mammal. In one embodiment, the formulation comprises a protein-cyclodextrin complex.